This invention relates to the generation and detection of genetic polymorphisms.
Genetic maps consisting primarily of restriction fragment length polymorphic (RFLP) markers are being constructed for many organisms, including man. Traditional approaches for detecting RFLPs involve Southern blot hybridization. Recently, techniques based on the polymerase chain reaction (PCR; Mullis et al., Methods Enzymol. 155:350-355, 1987) have been used in addition to, or in place of, traditional RFLP markers in genetic analysis (Cox et al., BioEssays 13:193-198, 1991). In contrast to traditional RFLP markers, PCR-generated markers can be scored using a small sample of DNA, without the use of radioactivity, and without the need for time-consuming DNA blotting procedures.
One widely used PCR-based approach involves the use of single short PCR primers of arbitrary sequence called RAPD primers (for random amplified polymorphic DNA; Reiter et al., Proc. Natl. Acad. Sci. USA 89:1477-1481, 1992; Williams et al., Theoret. Appl. Genet. 82:489-498, 1991). A second category of PCR-based markers are called SSLPs (for simple sequence length polymorphisms). The method employing SSLPs is based on amplification across tandem repeats of one or a few nucleotides known as "microsatellites." Microsatellites occur frequently and randomly in most eukaryotic genomes and display a high degree of polymorphism due to variation in the numbers of repeated units.
A third category of PCR-based markers are called AFLPs (for amplified fragment length polymorphisms). In the method employing these markers, DNA from two polymorphic strains are cleaved with one or two restriction endonucleases, and adapters are ligated to the ends of the cleaved fragments (Vos et al., Nucleic Acids Research 23: 4407-4414, 1995). The fragments are then amplified using primers complementary to the adapter(s). The primers contain short stretches of random nucleotides at their 3' ends, which result in limiting the number of amplified fragments generated.